Biochemical Characterization

15 May

Both types  of human 5a-reductases are hydrophobic, microsomal enzymes with  a molecular mass  of approximately 29 kDa  (Table  1). Type  1 isoenzyme, composed of 259 amino  acids,  has  an optimal pH  of 6.5 to 8.0, whereas type  2 isoenzyme is composed of 254 amino  acids  and  has an optimal pH  of 5.5 (Fig. 2). The determi- nants  of androgen binding are  encoded at the  ends  of the  molecule, while  those of  NADPH binding are  encoded in  the  carboxyl   terminal half  of  the  enzyme. Both  isoenzymes have  similar substrate preferences for  gene  structures (9). An average of 37% of the  amino  acids  have  side  chains  commonly found buried in the hydrophobic interior of globular proteins. These hydrophobic residues are dis- tributed throughout the enzyme and  do not  give rise to clear-cut transmembrane regions in hydropathy plots.  This structural feature suggests the 5a-reductase iso- enzymes are intrinsic membrane proteins deeply embedded in the lipid bilayer (15).

The enzymes have  never  been  fully  biochemically characterized (11), as the isolation and  purification have  been difficult  due  to the insolubility and,  generally, the  rather low  activity  of these  membrane-bound proteins. Since X-ray  patterns are  still  to  be  determined  (16),  only  indirect  information  is  available  on  the

TABLE 1    Properties of Human  Type 1 and Type 2 5a-Reductases

FIGURE 2    Activities  of human  recombinant type  1 and  type  2 5a-reductase expressed in CHO cells. The activities  were  determined over the indicated  pH range in the presence of 1 to 21.5 mM [14C]  testosterone  and   have   been  normalized to  the   highest  value   in  each  data   set.   They consequently are  represented as relative  enzyme activities.  O ¼ type 1 isoenzyme (50 pmol min21 mg21), B ¼ type 2 isoenzyme (15 pmol min21  mg21). Source: Adapted from Ref. 19.

three-dimensional structures of their binding sites. To circumvent these difficulties, expression-cloning strategies have  been used  to isolate  cDNA  (17).

Genetically, the type 1 isoenzyme is encoded by the SRD5A1 gene on the short arm of chromosome 5 (band  p15), while the type 2 isoenzyme by the SRD5A2 gene on chromosome 2 (band  p23). Both gene structures are similar in that each contains five exons  separated by four  introns (14).

Western blot studies of human cell cultures (sebocytes, keratinocytes, fibro- blasts,  dermal microvascular endothelial cells,  and  melanocytes) by  Chen  et  al. (8) show  two  closely  lying  bands of type  1 5a-reductase in  the  range  of 21 to

27 kDa,  possibly indicating the  existence  of heterogeneous proteins. This finding is supported by  a study of Lopez-Solache (18) that  reveals  the  presence of two forms  of mRNA  species  for rat  type  1 5a-reductase. Possible  explanations of the two bands could  be:

1.    partial protein denaturation during the process  of extraction or isolation,

2.    the  existence  of the  enzyme in  two  states,  activated or  inactivated, through either   phosphorylation, dephosphorylation (8), or  proteolytic cleavage of  a zymogen, and

3.    alternative splicing of mRNA.

Species Variation

Intraspecies and  interspecies comparisons of the  amino  acid  sequences between type 1 and type 2 isoenzymes show only moderate homologies, suggesting differing structure, functions, and/or regulations. Homology between the human type 1 and type  2 5a-reductases is approximately 50% (Fig. 3). The type  1 human and  rat iso- enzymes have approximately 61% amino  acid sequence identity, while the two cor- responding type  2 5a-reductases are 75% homologous (Table 1). The considerable differences of the enzymes concerning protein structure, functional characteristics,

FIGURE 3    Amino acid sequences of human  type 1 and type 2 5a-reductase. Shaded amino acids represent homologous items.  Asterisks above the  sequence stand for amino acid  positions  of naturally  occurring   mutants  in  type   2  isoenzyme  deficient   patients.  Hash   keys   above  the sequence denote the  tetrapeptide sequence implicated  in Finasteride  binding.  Source: Adapted from Ref. 19.

and  tissue  distribution would seem  to make  the  rat  a poor  choice  as a model  for comparative in  vivo  pharmacological assessment of novel  human 5a-reductase inhibitors. For example, rat  type  1 isoenzyme is at least  50-fold  more  susceptible to inhibition by Finasteride than  its human counterpart and  the activities of both type  1 and  type  2 5a-reductases can  be monitored in rat  prostate, while  activity of the type 2 isoenzyme predominates in tissue  from the mature human organ  (19).

In the Cynomolgus monkey, 5a-reductases are highly  homologous to their human  protein analogs, sharing about   93% (type  1) and  95% (type  2) amino acid   sequence  identity.  The   results  of  inhibition  studies  indicate  that   the monkey  isoenzymes  are  comparable on  a  molecular level  to  their   respective human  counterparts, supporting  the   relevance  and   use   of  the   Cynomolgus monkey  as  a  pharmacological  model   for  in  vivo  evaluation  of  5a-reductase inhibitors (19).

Enzymatic Activity and Specificity

5a-reductase, or more  precisely NADPH: 4-ene-3-oxosteroid 5-oxidoreductase (EC, is a NADPH-dependent enzyme that selectively and irreversibly catalyzes the  reduction of  the  4,5-double bond   of  4-ene-3-oxosteroids (e.g.,  testosterone,

FIGURE 4    (A) Proposed mechanism of the  catalysis of testosterone to DHT by 5a-reductase. (B) Substrate-like transition  state (Left) and  product-like  transition  state (Right). Source: Adapted from Ref. 16.

progesterone, and androstenedione) into the corresponding 5a-3-oxosteroids (DHT,

5a-dihydroprogesterone, and  5a-androstanedione) (13).

The  proposed mechanism (Fig.  4A)  of  testosterone reduction  to  DHT  by

5a-reductase  catalysis involves, as  a  key  step,  the  activation  of  the  4-en-3-on moiety  of testosterone by the interaction of the carbonyl group with an electrophilic residue Eþ  of the active site, followed by hydride transfer from NADPH to the pos- ition 5 (16). This leads  to enolate formation at C-3, C-4, which  presumably is stabil- ized by Eþ  at the active site. The process  may be viewed alternatively as activation of  the  enone  by  Eþ   leading to  a  positively polarized  species,  which   accepts  a hydride from  NADPH at C-5. Enzyme mediated tautomerism then  leads  to DHT with  release  of NADPþ  (5). Thus, it is possible to postulate two different transition states:  the “substrate-like” transition state in which  the C-5 has not yet changed its sp2  hybridization and  the  “product-like” transition state,  in  which   the  C-5 has assumed the final sp3 hybridization (Fig. 4B) (16).

The differences in primary sequences result  in unique functional character- istics of the two  isoenzymes. It has  been  proposed that  the  two  isoenzymes have different roles in androgen metabolism (13).

5a-reductases have apparent Km values  in the micro- to nanomolar range  for steroid substrates (Table 2) (20). The relative  affinity  of D4(5)-steroids for type 1 iso- enzyme is:

Note: Type 1 and type 2 5a-reductase activities were determined at pH 7.5 (50 mM sodium phosphate) and pH 5.0 (50 mM sodium citrate), respectively. 1 mM glucose-6-phosphate and 0.5 U/mL glucose-6-phosphate dehydrogenase was included  as a cofactor  regenerating system (NADPþ  to NADPH). Experiments were conducted using recombinant type 1 (0.05 – 0.10 mg protein  per assay) and  type 2 (0.02 – 0.15 mg protein  per assay) isoenzymes expressed in CHO cells. Standard errors  for all values were  less than  20% of the indicated  entries.

Source: Adapted from Ref. 19.

and  for the type  2 isoenzyme:

progesterone  ! 20a  hydroxyprogesterone  ! testosterone

! androstenedione  ! corticosterone

The  conversion  of  testosterone  to  DHT  amplifies  the  androgenic  signal through four  mechanisms:

1.    DHT,  unlike   testosterone, cannot be  aromatized to  estrogen, thus   its  effect remains purely androgenic (14).

2.    DHT binds to the human androgen receptor with a several-fold higher affinity than testosterone does (14). It has also been suggested that the DHT/androgen receptor complex has a higher affinity for the acceptor site in the nuclear chromatin (5).

3.    The DHT/androgen receptor complex appears to be more  stable  (14).

4.    Both   the   5a-reductase  genes   are   adjusted  by   DHT   via   a   feed-forward regulation (13).

Although testosterone and  DHT share  the  same  androgen receptor, it is not  sur- prising that  the two  androgens exert varying physiological effects.

Localization, Distribution, and Cutaneous Enzymatic Activity


The  specific  localization and  activity  of  type  1 isoenzyme in  sebaceous glands makes  this isoenzyme a potential therapeutic target  for the treatment of acne. For this  reason, it becomes important to understand where and  how  the  two  isoen- zymes  of 5a-reductase act in the skin (21).

Type 1 5a-reductase is present, above all, in the skin, whereas type 2 is predo- minantly located in the  prostate and  in genital  skin  (9). Both the  isoenzymes are expressed in the liver. In humans type 1 is not detectable in the fetus but is perma- nently  expressed in skin and  scalp  from  the time of puberty (13).

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